The phenotype of a cell is largely influenced by its display of integrins. By expressing several types of integrins on its surface, the cell is able to bind multiple kinds of ligands and thereby interpret parallel signals from the surrounding extracellular matrix (ECM). Cells cultured in vitro often express a different kind of integrin pattern than corresponding cells in vivo. In order to maintain the original phenotype of cells, or to accomplish a specific cellular response (e.g. differentiation, proliferation), it is important to enable integrin binding also during in vitro culture. This is most commonly done by coating cell culture plastics with ECM proteins like laminin, fibronectin, collagen or vitronectin, or mimics thereof. The ECM coatings will provide ligands for various integrins, with activation of different cellular pathways as a result. However, within several cell culture disciplines it is desirable to find ways to accomplish this on a defined matrix without the use of animal derived substrates.
WO 2011/129756 discloses methods and a cell scaffold material based on a miniature spider silk protein for eukaryotic cell culture. The protein may contain various short (3-5 amino acid residues) cell-binding peptides.
WO 2012/055854 discloses polymers consisting of a fusion protein containing a miniature spider silk protein and a large non-spidroin protein fragment of more than 30 amino acid residues which provides affinity to another molecule. The fusion protein may additionally contain various cell-binding peptides.
WO 2015/036619 discloses polymers consisting of a fusion protein containing a miniature spider silk protein and a cell-binding peptide comprising the amino acid residues RGD. The fusion protein is useful for cultivation of human pluripotent stem cells (hPSCs).
Several strategies have been attempted in order to accomplish ligands with high affinity and selectivity for specific integrins. For instance, phage libraries expressing RGD-containing peptides have been used in panning experiments. The outcome of such experiments is however dependent on limitations of the sequence coverage in the phage library. Moreover, epitopes that promote cell adherence might be missed when using a selection method that is based on inhibition of binding to coated integrins by peptides in solution. The interaction between a cell and the surrounding ECM is a cross-talk where initial binding causes intracellular signaling resulting in integrin activation and conformational changes that affects the affinity to the ligand. Thus, a cell-free system with coated integrins might miss the peptides with highest affinity to the activated form of the integrin. Ivanov, B. et al., Bioconjugate Chem. 6: 269-277 (1995) and Koivunen E. et al., Biotechnology 13(3): 265-270 (1995) disclose various RGD-containing peptides.
Several peptidomimetics and non-peptidic small molecules have been designed and synthesized with the purpose to find potent and selective integrin ligands. Rational design of ligands for certain integrins has been hampered by the lack of determined structures.
In most previous studies the goal has been to obtain a potent inhibitor of a specific integrin binding, for example with the purpose to hinder tumor cell invasion or unwanted angiogenesis. In those cases, a functional integrin binding is not required; rather the goal is a soluble molecule that is a potent integrin antagonist. WO 2013/185027 discloses soluble variants of human fibronectin with integrin antagonist activity, i.e. blocking or reducing activities of integrin, such as cell adhesion.
Despite these advances in the field, there is still a need for new cell scaffolds in the field, in particular since various cell types may have preference for different scaffolds and since there is a need for efficient cell scaffolds for wound healing.